How membrane-bound organelles of eukaryotic cells maintain their identity and subcellular localization amidst an enormous input and outflow of membrane and protein is a central question in cell biology. Studies in this group have focused on this question and have sought to define the cellular and molecular mechanisms which underlie the organization and distribution of eukaryotic organelles. Particular attention has been paid to the endoplasmic reticulum (ER)/Golgi membrane system, which plays a fundamental role in the biosynthesis, processing and sorting of protein moving through the secretory pathway, and the nuclear envelope (NE), which serves to compartmentalize the nucleus. Both the NE and Golgi complex are known to be highly dynamic structures and undergo complete disassembly and reassembly during mitosis. To further understand these processes and their relationship to mechanisms of organelle maintenance and trafficking, we have generated green fluorescent protein chimeras which localize to or traffic between the NE, Golgi complex or ER. Using these fusion proteins we have performed photobleaching recovery and time-lapse imaging experiments in living cells to analyze the steps involved in NE and Golgi complex disassembly/reassembly during mitosis. In addition, the fusion proteins have been used to characterize transport intermediates involved in secretory membrane traffic. Results from these studies are providing new insights into mechanisms of organelle targeting, maintenance and biogenesis.